BMI1 overexpression results in enhanced stem-cell frequencies and elevates their self-renewal potential. Cord blood CD34⁺ cells transduced with MiGR1 (open symbols) and BMI1 (closed symbols) were sorted in limiting dilutions and used to enumerate LTC-IC frequencies (A) or were injected into sublethally irradiated NOD-SCID mice (n = 5 per group; B). Human CD45⁺ chimerism levels were determined 6 weeks after transplantation in the BM of the transplanted mice (B). (C) After transduction, GFP⁺ CD34⁺CD38⁻ cells were sorted, cultured for 10 days in stroma-free cytokine-driven liquid culture conditions, and after which LTC-IC frequencies were determined on MS5 BM stroma in limiting dilution. (D) MiGR1 and BMI1-transduced CD34⁺ cells were cultured for 10 days in stroma-free conditions and 3.8 × 10⁶ cells were injected into sublethally irradiated NOD-SCID mice (n = 5 per group). Eight weeks after transplantation, human BM engraftment was evaluated on the basis of human CD45⁺ expression. (E) Transduced and sorted cord blood CD34⁺CD38⁻ cells were used to determine CAFC day 35 frequencies (left panel) and after 5 weeks the cultures were harvested and plated on new MS5 stroma to determine secondary LTC-IC frequencies (right panel). (F) Transduced CD34⁺ cells were used to perform transplantations into sublethally irradiated NOD-SCID mice (n = 5 per group) and engraftment levels after 8 weeks were determined (left panel). The BM from the primary recipients was used to perform secondary BM transplants and chimerism levels after an additional 8 weeks are shown in the right panel. (G) Multilineage engraftment of a representative mice transplanted with 10-day cultured cells is shown.